RESUMO
BRCA1 plays an important role in mechanisms of response to double-strand breaks, participating in genome surveillance, DNA repair, and cell cycle checkpoint arrests. Here, we identify a constitutive BRCA1-c-Abl complex and provide evidence for a direct interaction between the PXXP motif in the C terminus of BRCA1 and the SH3 domain of c-Abl. Following exposure to ionizing radiation (IR), the BRCA1-c-Abl complex is disrupted in an ATM-dependent manner, which correlates temporally with ATM-dependent phosphorylation of BRCA1 and ATM-dependent enhancement of the tyrosine kinase activity of c-Abl. The BRCA1-c-Abl interaction is affected by radiation-induced modification to both BRCA1 and c-Abl. We show that the C terminus of BRCA1 is phosphorylated by c-Abl in vitro. In vivo, BRCA1 is phosphorylated at tyrosine residues in an ATM-dependent, radiation-dependent manner. Tyrosine phosphorylation of BRCA1, however, is not required for the disruption of the BRCA1-c-Abl complex. BRCA1-mutated cells exhibit constitutively high c-Abl kinase activity that is not further increased on exposure to IR. We suggest a model in which BRCA1 acts in concert with ATM to regulate c-Abl tyrosine kinase activity.
Assuntos
Proteína BRCA1/metabolismo , Proteína BRCA1/efeitos da radiação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteína BRCA1/genética , Sítios de Ligação , Proteínas de Ciclo Celular , Células Cultivadas , Proteínas de Ligação a DNA , Humanos , Mutação , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/efeitos da radiação , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/efeitos da radiação , Radiação Ionizante , Proteínas Supressoras de TumorRESUMO
We have generated transgenic mice that harbor a 140 kb genomic fragment of the human BRCA1 locus (TgN x BRCA1GEN). We find that the transgene directs appropriate expression of human BRCA1 transcripts in multiple mouse tissues, and that human BRCA1 protein is expressed and stabilized following exposure to DNA damage. Such mice are completely normal, with no overt signs of BRCA1 toxicity commonly observed when BRCA1 is expressed from heterologous promoters. Most importantly, however, the transgene rescues the otherwise lethal phenotype associated with the targeted hypomorphic allele (Brca1DeltaexllSA). Brca1-/-; TgN x BRCA1GEN bigenic animals develop normally and can be maintained as a distinct line. These results show that a 140 kb fragment of chromosome 17 contains all elements necessary for the correct expression, localization, and function of the BRCA1 protein. Further, the model provides evidence that function and regulation of the human BRCA1 gene can be studied and manipulated in a genetically tractable mammalian system. Oncogene (2000) 19, 4085 - 4090
Assuntos
Proteína BRCA1/genética , Perda do Embrião/genética , Técnicas de Transferência de Genes , Animais , Proteína BRCA1/metabolismo , Proteína BRCA1/efeitos da radiação , Northern Blotting , Southern Blotting , Linhagem Celular , Dano ao DNA/genética , Raios gama , Vetores Genéticos , Humanos , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos , Fenótipo , Regiões Promotoras GenéticasRESUMO
Camptothecin (CPT) and X-ray (XR) generate double-strand breaks (DSB) that can be processed by homologous or nonhomologous recombination. We studied the participation of proteins involved in recombination pathways and cell cycle control in the signal transduction between DNA damage and NF-kappaB. Cells harbouring mutated NBS, hMRE11, BRCA1 or MLH1 were analysed. NBS- and hMRE11-deficient cells present a classical kinetic of NF-kappaB induction after camptothecin treatment. When DSB are generated by XR, NBS-deficient cells exhibit a delayed and strongly reduced level of NF-kappaB induction, whereas the hMRE11 mutated cells do not induce NF-kappaB at all. This indicates an important role of the hMRE11/hRAD50/NBS complex in the signal transduction initiated by XR. In HCC1937 cells that express a truncated version of BRCA1, XR induces a very rapid and transient NF-kappaB activation, whereas CPT leads to a delayed activation suggesting that BRCA1 modulates the transduction pathways in different manners after these two stresses. Finally, we found that a proficient MMR pathway is essential to the NF-kappaB activation after both CPT and XR. These results indicate that DSB originating from XR or CPT do not induce NF-kappaB in a unique way. MMR participates in both cascades, whereas the hMRE11/hRAD50/NBS trimer is specifically involved in the response elicited by XR.
Assuntos
Proteína BRCA1/metabolismo , Camptotecina/farmacologia , Enzimas Reparadoras do DNA , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Raios X , Hidrolases Anidrido Ácido , Proteínas Adaptadoras de Transdução de Sinal , Proteína BRCA1/efeitos dos fármacos , Proteína BRCA1/genética , Proteína BRCA1/efeitos da radiação , Proteínas de Transporte , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/efeitos da radiação , DNA/efeitos dos fármacos , DNA/efeitos da radiação , Dano ao DNA/fisiologia , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/efeitos da radiação , Inibidores Enzimáticos/farmacologia , Células HeLa/efeitos dos fármacos , Células HeLa/efeitos da radiação , Humanos , Proteína Homóloga a MRE11 , Substâncias Macromoleculares , Proteína 1 Homóloga a MutL , Mutação , NF-kappa B/efeitos dos fármacos , NF-kappa B/efeitos da radiação , Proteínas de Neoplasias/efeitos dos fármacos , Proteínas de Neoplasias/efeitos da radiação , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/efeitos da radiação , Radiação Ionizante , Transdução de SinaisRESUMO
BACKGROUND: In recent years data from both mouse models and human tumors suggest that loss of one allele of genes involved in DNA repair pathways may play a central role in genomic instability and carcinogenesis. Additionally several examples in mouse models confirmed that loss of one allele of two functionally related genes may have an additive effect on tumor development. To understand some of the mechanisms involved, we examined the role of monoallelic loss or Atm and Brca1 on cell transformation and apoptosis induced by radiation. METHODS: Cell transformation and apoptosis were measured in mouse embryo fibroblasts (MEF) and thymocytes respectively. Combinations of wild type and hemizygous genotypes for ATM and BRCA1 were tested in various comparisons. RESULTS: Haploinsufficiency of either ATM or BRCA1 resulted in an increase in the incidence of radiation-induced transformation of MEF and a corresponding decrease in the proportion of thymocytes dying an apoptotic death, compared with cells from wild-type animals. Combined haploinsufficiency for both genes resulted in an even larger effect on apoptosis. CONCLUSIONS: Under stress, the efficiency and capacity for DNA repair mediated by the ATM/BRCA1 cell signalling network depends on the expression levels of both proteins.
Assuntos
Desequilíbrio Alélico , Apoptose/genética , Proteína BRCA1/genética , Proteínas de Ciclo Celular/genética , Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/genética , Desequilíbrio Alélico/efeitos da radiação , Animais , Apoptose/efeitos da radiação , Proteínas Mutadas de Ataxia Telangiectasia , Proteína BRCA1/efeitos da radiação , Proteínas de Ciclo Celular/efeitos da radiação , Transformação Celular Neoplásica/efeitos da radiação , Ensaio Cometa , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/efeitos da radiação , Camundongos , Proteínas Serina-Treonina Quinases/efeitos da radiação , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiação , Proteínas Supressoras de Tumor/efeitos da radiaçãoRESUMO
Recent studies have provided evidence that breast cancer susceptibility gene products (Brca1 and Brca2) suppress cancer, at least in part, by participating in DNA damage signaling and DNA repair. Brca1 is hyperphosphorylated in response to DNA damage and co-localizes with Rad51, a protein involved in homologous-recombination, and Nbs1.Mre11.Rad50, a complex required for both homologous-recombination and nonhomologous end joining repair of damaged DNA. Here, we report that there is a qualitative difference in the phosphorylation states of Brca1 between ionizing radiation (IR) and UV radiation. Brca1 is phosphorylated at Ser-1423 and Ser-1524 after IR and UV; however, Ser-1387 is specifically phosphorylated after IR, and Ser-1457 is predominantly phosphorylated after UV. These results suggest that different types of DNA-damaging agents might signal to Brca1 in different ways. We also provide evidence that the rapid phosphorylation of Brca1 at Ser-1423 and Ser-1524 after IR (but not after UV) is largely ataxia telangiectasia mutated (ATM) kinase-dependent. The overexpression of catalytically inactive ATM and Rad3 related (ATR) kinase inhibited the UV-induced phosphorylation of Brca1 at these sites, indicating that ATR controls Brca1 phosphorylation in vivo after the exposure of cells to UV light. Moreover, ATR associates with Brca1; ATR and Brca1 foci co-localize both in cells synchronized in S phase and after exposure of cells to DNA-damaging agents. ATR can itself phosphorylate the region of Brca1 phosphorylated by ATM (Ser-Gln cluster in the C terminus of Brca1, amino acids 1241-1530). However, there are additional uncharacterized ATR phosphorylation site(s) between residues 521 and 757 of Brca1. Taken together, our results support a model in which ATM and ATR act in parallel but somewhat overlapping pathways of DNA damage signaling but respond primarily to different types of DNA lesion.
Assuntos
Proteína BRCA1/metabolismo , Proteína BRCA1/efeitos da radiação , Proteínas de Ciclo Celular , Proteínas Serina-Treonina Quinases/metabolismo , Raios Ultravioleta , Ataxia Telangiectasia , Proteínas Mutadas de Ataxia Telangiectasia , Proteína BRCA1/química , Linhagem Celular , Proteínas de Ligação a DNA , Raios gama , Humanos , Cinética , Fosforilação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Proteínas Supressoras de TumorRESUMO
The tumour-suppressor protein BRCA1 mediates its biological functions by interacting with cellular factors such as the CtIP polypeptide, a substrate for the ATM (for 'ataxia telangiectasia mutated') protein kinase. Li et al. report that the BRCA1-CtIP interaction is disrupted by ionizing radiation and by other genotoxic stresses that induce phosphorylation of CtIP by ATM kinase, and that this dissociation of the BRCA1-CtIP complex in turn modulates the transcription of DNA-damage-response genes. We have shown that the BRCA1-binding domain of CtIP (amino-acid residues 133-369) is distal to the sites that are phosphorylated by ATM kinase (residues S664 and S745). We now show that the BRCA1-CtIP complex is stable in irradiated cells, and that the phosphorylated isoforms of CtIP that are induced by ionizing radiation still interact in vivo with BRCA1. We conclude that disruption of the BRCA1-CtIP complex cannot account for induction of DNA-damage-response genes in the way proposed by Li et al.
Assuntos
Proteína BRCA1/metabolismo , Proteínas de Transporte/metabolismo , Dano ao DNA , Proteínas Nucleares/metabolismo , Anticorpos Monoclonais , Proteínas Mutadas de Ataxia Telangiectasia , Proteína BRCA1/efeitos da radiação , Proteínas de Transporte/efeitos da radiação , Proteínas de Ciclo Celular , Linhagem Celular Transformada , DNA/metabolismo , DNA/efeitos da radiação , Dano ao DNA/genética , Proteínas de Ligação a DNA , Endodesoxirribonucleases , Humanos , Proteínas Nucleares/efeitos da radiação , Fosforilação , Testes de Precipitina , Ligação Proteica , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/efeitos da radiação , Proteínas Serina-Treonina Quinases/metabolismo , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor , Raios UltravioletaRESUMO
To investigate changes in BRCA1 following DNA damage, we exposed MCF-7 cells to increasing doses of ultraviolet C. We observed an increase in BRCA1 protein levels above 78 J/m2. This increase was observed as early as 5 min after irradiation. BRCA1 levels were then observed to decrease after 2 h, consistent with the previously published data. By pretreating with cycloheximide prior to irradiation, we observed a decrease in the protein half-life, from 3.5 h to 53 min, suggesting that a decrease in protein half-life may cause the lower levels of BRCA1 after irradiation. We also observed an increase in BRCA1 mRNA within 15 min of irradiation, followed by a decrease after 4 h. These data suggest that newly translated protein may contribute to increases in BRCA1 protein levels. The very rapid changes in BRCA1 support its role as a sensor of DNA damage, as opposed to being a repair gene.
Assuntos
Proteína BRCA1/genética , Proteína BRCA1/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Genes Neoplásicos/efeitos da radiação , Proteína BRCA1/metabolismo , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Cicloeximida/farmacologia , Dano ao DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Epitopos , Feminino , Citometria de Fluxo , Meia-Vida , Humanos , Cinética , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Fase S/efeitos da radiação , Células Tumorais Cultivadas , Raios UltravioletaRESUMO
Germ-line mutations of the BRCA1 and BRCA2 genes predispose women to develop cancers of the breast and ovary, but the biologic functions of these genes remains unclear. We have investigated the responses of the BRCA1 and BRCA2 gene products to cytotoxic agents in 3 human ovarian cancer cell lines: SK-OV-3 (which contains a p53 deletion mutation), CAOV-3 (which over-expresses a mutant p53) and PA-1 (which expresses wild-type p53). In screening studies, we determined the effects of 7 different agents on BRCA1 and BRCA2 expression. We found that Adriamycin (ADR) and ultraviolet (UV)radiation significantly down-regulated BRCA1 and BRCA2 mRNA expression in SK-OV-3 cells. On the other hand, camptothecin, nitrogen mustard, taxol, vincristine and etoposide had no effect on BRCA1 or BRCA2 mRNA levels at doses that yielded degrees of cytotoxicity similar to or greater than ADR. The down-regulation of BRCA1 and BRCA2 mRNAs was dose and time dependent; significant down-regulation was first observed at 8-16 hr after exposure to ADR. BRCA1 protein levels were also down-regulated following treatment of SK-OV-3 cells with ADR. Similar results were observed in CAOV-3 and PA-1 cells treated with ADR, and this finding could not be directly attributed to ADR-induced changes in the cell cycle distribution. The ADR doses required for significant decreases of BRCA1 and BRCA2 were about 10-15, 5-10 and 2 microM, respectively, for SK-OV-3, CAOV-3 and PA-1; the IC50 doses for loss of cell viability (determined by Trypan blue dye exclusion) were 23, 14 and 0.4 microM, respectively. Thus, at equitoxic doses of ADR, PA-1 cells were more resistant to down-regulation of BRCA1 and BRCA2 than SK-OV-3 or CAOV-3. Our findings suggest that 1) BRCA1 and BRCA2 expression in human ovarian cancer cell lines is selectively down-regulated by 2 DNA-damaging agents (ADR and UV radiation); 2) these responses are not due to non-specific cytotoxicity; and 3) the BRCA1 and BRCA2 responses may be dependent, in part, on the p53 functional status of the cells. We speculate that the down-regulation of BRCA1 and BRCA2 may be part of a cellular survival response activated by certain forms of DNA damage.